1. Field of the Invention
The present invention relates to the detection and quantitative measurement of proteins.
2. Description of the Prior Art
Several methods are known for detecting the presence of protein in samples. These include the classical Lowry (Lowry, Oh. H., Rosebrough, N.J., Farr, A. L., and Randall R. J. (1951), J. Biol. Chem. 193, 265-275.) and biuret (Mokrasch, L. C., and McGilvery, R. W. (1956) J. Biol. Chem. 221, 909-917) methods which suffer from interference by many species commonly present in protein-containing solutions. Moreover, modified procedures for eliminating such problems are also disadvantageous since they are complicated and time consuming.
Another general protein analysis technique involves detection of a color change which occurs when a dye such as Orange G or bromo cresol green or purple becomes bound to a protein. These methods are based on the protein error phenomenon whereby proper buffering of the dye-containing reagent enables the normal pH sensitive color change of the dye to occur upon binding to the protein with no pH change. These methods are quite insensitive, detecting from mg to g of proteins; are only applicable to a limited number of proteins, typically to only a single one such as albumin, and are not applicable to protein quantitation in many important biological fluids such as urine. Moreover, for the dye binding technique, satisfactory sensitivity is achievable only by elimination of turbidity from the test mixture, for example by addition of surfactants (U.S. Pat. No. 3,884,637) and/or sequestering agents (U.S. Pat. No. 3,872,272) to prevent the precipitation of the dye/protein complex. A more sensitive dye binding technique does exist, but it requires precipitation and filtration and is therefore complicated and time consuming (Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56, 502-514).
Dye/protein complex formation is also utilized for staining of proteins in gels used in electrophoresis. For example, the dye Coomassie Brilliant Blue G-250 in perchloric acid solution has been so used (Reisner, A. H. et al (1975) Anal. Biochem. 64, 509-516). However, these techniques involve precipitation of the colored protein/dye complex and are inapplicable to sensitive quantitative analysis of protein-containing solutions where spectrophotometric means are generally used for measurements. Consequently, a need exists for a more sensitive and rapid method for quantitative analysis of small amounts (.mu.g) of protein.